Biosynthesis of secondary metabolites in sugarcane

A set of genes related to secondary metabolism was extracted from the sugarcane expressed sequence tag (SUCEST) database and was used to investigate both the gene expression pattern of key enzymes regulating the main biosynthetic secondary metabolism pathways and the major classes of metabolites involved in the response of sugarcane to environmental and developmental cues. The SUCEST database was constructed with tissues in different physiological conditions which had been collected under varied situation of environmental stress. This database allows researchers to identify and characterize the expressed genes of a wide range of putative enzymes able to catalyze steps in the phenylpropanoid, isoprenoid and other pathways of the special metabolic mechanisms involved in the response of sugarcane to environmental changes. Our results show that sugarcane cDNAs encoded putative ultra-violet induced sesquiterpene cyclases (SC); chalcone synthase (CHS), the first enzyme in the pathway branch for flavonoid biosynthesis; isoflavone synthase (IFS), involved in plant defense and root nodulation; isoflavone reductase (IFR), a key enzyme in phenylpropanoid phytoalexin biosynthesis; and caffeic acid-O-methyltransferase, a key enzyme in the biosynthesis of lignin cell wall precursors. High levels of CHS transcripts from plantlets infected with Herbaspirillum rubri or Gluconacetobacter diazotroficans suggests that agents of biotic stress can elicit flavonoid biosynthesis in sugarcane. From this data we have predicted the profile of isoprenoid and phenylpropanoid metabolism in sugarcane and pointed the branches of secondary metabolism activated during tissue-specific stages of development and the adaptive response of sugarcane to agents of biotic and abiotic stress, although our assignment of enzyme function should be confirmed by careful biochemical and genetic supporting evidence.


INTRODUCTION
Plants interact with their environment by producing a diverse array of secondary metabolites (Harborne, 1996).Many of these compounds are valued for their pharmacological activities and industrial or agricultural properties which increase the commercial value of crops (Paganga et al., 1999;Bingham et al., 1998).
There is an emerging body of evidence, based on biological activities in vitro and on the correlation between rates of accumulation and expression of resistance in vivo, which indicates that isoprenoid and phenylpropanoid compounds play a role in the induced and constitutive response of plants to biotic and abiotic agents (Dixon and Paiva,1995).
Although the isoprenoid pathway has, for the most part, been described in animals and fungi and many enzymes involved in various steps of isoprenoid biosynthesis have been measured in plants systems, a variety of essential and developmentally regulated isoprenoids are synthesized in plants but not in other eukaryotes and it remains to be validated whether or not this pathway is operative in plants.
Phenylpropanoids are involved in several functions and play a vital role in the interaction between plants and their environment.Many aromatic secondary metabolites are phenylpropanoids, e.g.flavonoids, phytoalexins, agents which protect against ultra-violet light and, most importantly, lignin (Mabry and Ulubelen,1980).Compounds that are constitutive in one plant species or tissue can be induced by various stresses in another species or in another tissue of the same plant (Beggs et al.,1987;Christie et al., 1994).Expression of the phenylpropanoid biosynthetic pathway is precisely regulated in response to developmental signals, nutrient status and environmental stimuli such as light, heat and pathogen attack.The accumulation of flavonoids and isoflavonoids under the attack of pathogens is used as a mechanism of detoxification by many species (Van Etten et al.,1989).
In most systems studied, the induction of isoprenoid and phenylpropanoid synthesis under conditions of stress is the result of increased transcription of genes encoding the corresponding biosynthetic enzymes (Alex et al., 2000;He et al., 1998;Fahrendorf and Dixon, 1993;Uhlmann and Ebel,1993).
Recombinant DNA technology offers new opportunities to directly modify plant secondary product synthesis through metabolic engineering, and the availability of cloned genes encoding terminal enzymes of isoprenoid or phenylpropanoid compounds may provide the means to assess product function by using reverse genetics (i.e.anti sense or sense suppression) in species that are amenable to genetic transformation.However, despite the increasing number of proteins isolated from plant species there is a lack of genes encoding key secondary metabolism enzymes.
The SUCEST database was constructed with tissues in different physiological conditions which had been developed under varied situation of environmental stress and represents a rich source from which to identify and characterize expressed genes of a wide range of putative enzymes able to catalyze steps in the phenylpropanoid, isoprenoid and other pathways of the special metabolic mechanisms related to the response of sugarcane to environmental changes.In addition, because the protein structure of most enzymes involved in secondary metabolism is conserved, cloned genes from sugarcane may be useful as probes for the isolation of related genes from other monocotyledon (or even dicotyledon) species.
The work described in this paper was carried out to produce a collection of genes related to secondary metabolism and also to investigate the gene expression pattern of key enzymes which regulate the main biosynthetic routes of secondary metabolism, uncovering the major classes of metabolites that play a role in the response of sugarcane to environmental and developmental cues.

Source library
The source tissues, sugarcane variety and the codes used in this paper are shown in Table I.

Isoprenoid pathway enzymes
In the main trunk of the isoprenoid pathway the enzymes squalene synthetase (SQS -EC 2.5.1.21)(Porter and Spurgeon, 1981) and sesquiterpene cyclase (SC -EC 2.1.5.) (Cane,1981) are the first branch point enzymes for sterol and sesquiterpenoid biosynthesis competing for the last common intermediate between the two branches, farnesyl diphosphate (FPP).FPP is also a precursor for the biosynthesis of other polyprenyl-lipids and isoprenoids including carotenoids, gibberellic acid, ubiquinones and dolichols (Porter and Spurgeon, 1981).Cycloartenol synthase (CAS -EC 5.4.99.8), another enzyme involved in isoprenoid metabolism, converts oxidosqualene to cycloartenol, the first carbocyclic intermediate in the route to sterols in plants.
A cDNA clone (GgCAS1) encoding CAS was isolated from Glycyrrhiza glabra (licorice) by cross-hybridization with a probe of Pisum sativum CAS.The deduced amino acid sequence of GgCAS1 exhibited 89%, 83% and 81% identity to P. sativum, Panax ginseng and Arabidopsis thaliana CASs, respectively (Husselstein-Muller et al., 2001).Protein sequences from these plants were used as virtual probe to search for CAS genes in the SUCEST database.
A search of the SUCEST database, using the basic local alignment search tool (BLAST) (Altschul et al., 1990), with proteins previously isolated from other plant species led us to identify DNA sequences with high homology to catalytic proteins of the isoprenoid and phenylpropanoid metabolism pathway.These clones were sequenced and when the putative products were compared to the published amino acid sequences for the corresponding enzymes the best percentages of similarity ranged from 65% to 93%.Multiple alignment, using the Clustal X program of Jeanmougin et al. (1998), of full length sequence of sugarcane with similar proteins showed the transcripts which are closely related to the protein structure of each key enzyme of the main branches of the isoprenoid and phenylpropanoid pathways in plants.

Sesquiterpene cyclase
The abundance of sugarcane expressed sequence tags (ESTs) and putative isoprenoid and phenylpropanoid related enzymes is shown in Table II, abundance being shown as the number of enzyme related transcripts divided by the number of ESTs in the SUCEST library.
Comparative analysis between the deduced amino acid sequences from the SUCEST database and genes coding for sesquiterpene cyclase (SC) showed that the SCSBFL1107C10 sequence is 44% related to the a (+)-delta-cadinene synthase gene gi:8389329 which is involved in the biosynthesis of sesquiterpene aldehydes in the plant Gossypium arboreum L. (Tan et al., 2000) and that the SCCCFL4O89E11 sequence has a similarity of 45% in relation to an ultra-violet induced sesquiterpene cyclase gene gi: 6644409 from Capsicum annum.In addition, sequence SCEQAD1018E06 has a 44% resemblance to the gene gi: 5360687 which codes for a vetispiradiene synthase expressed in potato tubers infected with Phytophthora infestans (Yoshioka et al., 1999).Based on the abundance and type of transcripts identified we suggest that sugarcane flower and leaf tissues express sesquiterpene cyclases from the TPSa subgroup of angiosperms.

Squalene synthases
A search of the SUCEST database with genes encoding squalene synthases (SQS) from Oryza sativa (gi:2463565) Zea mays (gi: 7434086) and P. ginseng (gi:2780361) showed putative squalene synthase cDNAs of which the best match was the sequence SCCCRT2002B10 which shared 93% identity with the gi:7434086 gene from Z. mays.Multiple sequence alignment between SQS-related sequences from sugarcane, monocotyledons and dicotyledons reinforce the assumption made by Hata et al.For library key see Table I.
(1997) that SQS from monocotyledons and dicotyledons form distinct evolutionary subgroups.
Since they are key enzymes at branch points of the main trunk of the isoprenoid pathway the putatively identified squalene synthase and sesquiterpene cyclase genes may control the level of expression of different isoprenoid classes in sugarcane tissues.The expression pattern of SQS and CAS in the AD tissue-library indicates that the branch for sterol biosynthesis is active in plantlets infected with Gluconacetobacter diazotroficans , suggesting that sugarcane produces phytosterols in response to this bacteria.(Table III).On the other hand, the expression of the phenylpropanoid pathway enzymes IFS, CHS and CHI in plantlets infected with Herbaspirillum rubri (HR tissue-library) showed that different biosynthetic branches were activated (Table III).
In cell cultures of Nicotiana tabacum treated with a fungal elicitor, biotic elicitation can also cause coordinated inhibition of squalene synthase and induction of sesquiterpene cyclase resulting in a rapid decrease in phytosterol biosynthesis coincident with the induction and accumulation of novel terpenoids involved in the plant defense response (Threlfall and Whitehead, 1988;Voegeli and Chappell, 1988).
In the cDNA libraries constructed with various tissues of sugarcane variety SP 80-3280, comparison of the gene expression pattern related to the isoprenoid pathway indicated that stem bark (SB-library) and lateral buds (LB-library) are probable sites of biosynthesis of terpenoid and steroid compounds.Stem bark tissue also expressed genes related to the biosynthesis of lignin precursors and isoflavonoid compounds.In addition, high level expression of phenylpropanoid genes was detected in first and fourth internode tissues (ST-library) which suggests that metabolic routes for the biosynthesis of more complex phenylpropanoids are active in this tissue.Rolled leaves seem to be the site for the synthesis of flavonone-like metabolites.Moreover, in root tissue, high levels of CHS and C4H (which regulates the biosynthesis of the substrate for chalcone synthase) indicate that products such as tetrahydrochalcone may accumulate in this tissue (Table IV).

Phenylpropanoid metabolism Chalcone synthase and chalcone isomerase
Sequences matching chalcone synthase (CHS) and chalcone isomerase (CHI) (enzymes committed to the central branch of the flavonoid biosynthesis pathway) were detected in the SUCEST database.The clones found were full-length sequenced and the transcripts compared to corresponding genes from other plant species.It was found that the sugarcane CHS protein sequences were similar to the sorghum genes encoding proteins CHS 1 (gi: 5005907), CHS 2 (gi: 5005909) and CHS 3 (gi: 5005911) as well as to the Maize gene gi: 116380 which also shared high identity with the sugarcane sequence SCEZRT2018G01 (Figure 2).

Isoflavone synthase
Isoflavone synthase (IFS) catalyzes the first committed step of isoflavone biosynthesis, a branch of the phenylpropanoid pathway.Isoflavones have received much  For library key see Table I.
attention because of their benefits to human health.These compounds are thought to be produced almost exclusively in legumes and play natural roles in plant defense and root nodulation.Genes involved in isoflavone biosynthesis were isolated from sugarcane, sequenced and the putative products showed similarities in their sequences to IFS genes from sorghum (gi: 5915857, 97% similarity) and A. thaliana (gi: 4895205 (70%) and gi: 5915859 (69%)).
Isoflavone reductase Isoflavone reductase (IFR) is an enzyme specific to isoflavonoid biosynthesis, a pathway so far only found in legumes.It catalyses a NADPH-dependent reduction involved in the biosynthesis of important and related phenylpropanoid-derived plant defense compounds.IFR has been cloned from alfalfa and pea, and it has been demonstrated that when IFR is expressed in Escherichia coli it has the same substrate and product specificity as the purified native plant enzyme (Paiva et al.,1991(Paiva et al., ,1994)).In our work we isolated three related IFR-like sugarcane cDNAs, sequence SCVPLB1020E05 similar to the Z. mays gene gi: 1708421 which encodes isoflavone reductase (a NADPH-binding enzyme activated in response to sulfur starvation), the SCEZRZ1016H12 sequence with homology to the Lupinus albus gene gi: 1708424 which codes for an isoflavone reductase-like protein and the SCJFRZ2010G10 sequence which shares identity with the A. thaliana gene gi: 7484777 (Figure 3).

O-methyltransferases
Our search of the SUCEST database revealed sequences with similarities to O-methyltransferases (OMT) from various sources and classes.The highest matches (97-100% identity) were found between the gi: 3341509 caffeoyl 3-O-methyltransferase (involved in lignification) gene from Saccharum officinarum and the sugarcane expressed sequence transcripts SCQSRT2031E06, SCCCRT 1003A12, SCRFLR1012F12 and SCVPRT2073D10.Expressed sequences from apical meristem (AM-library) were    C For library key see Table I.
94% identical to the caffeoyl 3-O-methyltransferase gene gi: 5101868 from Z. mays.The putative protein SCQGRT1039F03 was 59% similar to the gi:1353193 gene encoding maize OMT (ZRP4) and may be involved in the O-methylation of suberin phenylpropanoid precursors, while root-tissue (RT-library) OMT-type expressed sequences showed 51% homology with a herbicide safener-binding protein, antidote used to protect crops from herbicide injury, from Z. mays (gi:7447882).The relative abundance of the identified transcripts indicated that putative OMT proteins from sugarcane accumulate preferentially in the roots, because we detected low frequencies of these transcripts in leaf, stem and other aerial tissues.

DISCUSSION
Several phenylpropanoids (e.g.coumaric acid and simple coumarins) are produced from cinnamate via a series of hydroxylation, methylation and dehydration reactions.A large number of phenylpropanoids are derived from the C15 flavonoid skeleton which is synthesized via the CHS-catalyzed condensation of p-coumaroyl-coenzyme A and three molecules of malonyl-CoA (Harborne, 1988).In most plant families, the initial product of CHS is a tetrahydroxychalcone which is further converted to other flavonoid classes (Holton and Cornish, 1995).When both CHS and CHR are expressed, as in legumes, a trihydroxychalcone is formed and further transformed to its isomer by the action of CHI (Welle and Grisebach, 1989).
The abundance of clones for putative enzymes of both the central phenylpropanoid pathway and specific branch pathways of isoflavonoid or furanocoumarin synthesis in each source library of the SUCEST database suggest that CHS is active in root-tissue (RT-library) where it catalyzes the biosynthesis of chalcones which may be converted to flavonoid derivatives by CHI in rolled-leaves (LR-library) and stem-tissue from the first and fourth internodes (ST-library) where, in the presence of IFS and IFR, the flavonoid skeleton can probably be re-arranged leading to the accumulation of various simple isoflavonoids or more complex flavonoid derivatives.Ultra-violet radiation induces flavonoids (particularly kaempferol derivatives) and sinapate esters in A. thaliana and isoflavonoids and psoralens (furanocoumarin) in other species.These UV-absorbing compounds are thought to protect against the damage and subsequent cell death caused by UV-B by protecting DNA from dimerization and breakage (Beggs et al., 1985;Lois, 1994).
In healthy tissues the highest level of CHS transcripts were detected in roots, consistent with the biosynthesis of flavonoid derivatives in these tissues (Table II).The high frequency of CHS transcripts from plantlets infected with H. rubri or G. diazotroficans suggests that biotic stress agents cause elicitation of flavonoid biosynthesis in sugarcane species (Table III).
In our search we found sugarcane cDNA sequences with similarity to a Forsythia intermedia gene encoding (+)-pinoresinol/(+)-lariciresinol reductase, which, according to Dinkova-Kostova et al. (1996), regulates a pivotal branch point in lignan biosynthesis.The amino acid sequence of this enzyme reveals a strong homology to isoflavone reductase, a key branch point enzyme in isoflavonoid metabolism which is mainly found in the Leguminosae (angiosperms).Lignans are a widely distributed class of natural products, whose functions and distribution suggest that they are one of the earliest forms of defense mechanism to have evolved in vascular plants, whereas the isoflavonoids produced by isoflavone-reductase are mainly restricted to the Leguminosae.In fact, further evidence as to which specific class of secondary compounds sugarcane defense products belong would contribute to the understanding of the evolution of plant defense mechanisms.
Metabolic engineering for the production of isoflavones in non-legume dicotyledon and monocotyledon tissues may provide additional human health benefits due to the more wide consumption of grains containing these phyto-oestrogens (Bingham et al., 1998).It has been proved that in legumes, isoflavones act in both the symbiotic relationship with rhizobial bacteria and the plant defense response (Phillips and Kapulnik, 1995;Savoure et al.,1994).
Although, the accumulation of indole alkaloid metabolites in sugarcane has not been so far reported, the unexpected finding of putative strictosidine synthase (Str) genes in the flower, root and leaf-roll tissue cDNA libraries suggests that the indole pathway is active in sugarcane.The amino acid deduced sequence for the SCUTFL1064 sequence had an 83% similarity to the gi:5777623 gene which codes for Str in Oryza sativa.
Although the assignment of enzyme function must be confirmed by further biochemical and/or genetic evidence, this work has allowed us to anticipate the isoprenoid and phenylpropanoid metabolism profile of sugarcane.It has also indicated the branch pathways of secondary metabolism activated during different tissue-specific developmental stages and under adaptive response to biotic and abiotic stress agents.
The information on expression patterns within the various sugarcane tissues provided in this paper can be used to handle the production of a specific metabolite within the plant using biochemical and genetic manipulation of secondary metabolism, which up to now has mainly been studied in medicinal plants at the final product level.The increasing amount of data on genome sequences from different plant species is changing our view of how widespread these pathways are in the plant kingdom, bringing us to the question `is every plant a potential medicinal plant?'.Genomics is opening a `molecular avenue' to the study of plant secondary metabolism which will enable us to study the genes coding for the key metabolic enzymes, their mo-lecular regulation and ecological and physiological roles as well the evolutionary significance of the pathway involved.This connection between the phytochemistry of natural products and genomics will allow the exploration of a wide range of plants for biotechnological purposes.

Figure 3 -
Figure 3 -Cladogram of cDNAs coding for putative isoflavone reducthases and corresponding IFR genes from other plants.* Multiple alignment analysis was performed with Clustal X program (www.igbmc.u-strasbg.fr).The length of the horizontal lines indicates an inverse degree of relatedness.gi:7484777 Arabidopsis; gi:1708424 Lupinus albus; gi:1708421 Zea mays.

Table I -
Library codes of the sugarcane source tissues and varieties used in this study.
a Grown in vitro.b Grown in a green house at Centro de Biologia Molecular e Engenharia Genética (CBMEG) Universidade Estadual de Campinas.C.P. 6109, Campinas, SP, Brazil.c Grown at the experimental station of Cooperativa de Produtores de Açucar e Alcool (COPERSUCAR), Piracicaba, SP, Brazil.d Collected at the experimental station of the Universidade Federal de Alagoas (UFAL) Muricí, Al, Brazil.For more details on library source see: http://sucest.lad.ic.unicamp.br.

Table II -
Abundance of sugarcane expressed sequence tags (ESTs) and putative isoprenoid and phenylpropanoid related enzymes.Abundance is shown as the number of enzyme related transcripts divided by the number ESTs in the library.

Table III -
Gene expression pattern of tissues from sugarcane variety SP 70 -1143.Values represent the ratio of enzyme related transcript abundance (x 10 5 ) multiplied by the total reads of the library in the SUCEST database.

Table IV -
Gene expression pattern of tissues from sugarcane variety SP 80 -3280.Values represent the ratio of enzyme related transcript abundance (x 10 5 ) multiplied by the total reads of the library in the SUCEST database.